Since its introduction in 1999, West Nile virus (WNV) has become the leading cause of arboviral encephalitis in the United States. The most notable epidemiological factors associated with the rapid emergence of WNV in North America have been the development of high viral titers within birds and mortality among wild bird populations. Despite the use of crow mortality as a sentinel for WNV activity, the underlying viral genetic basis for WNV pathogenicity in birds and the role of increased avian virulence in the transmission of WNV are poorly understood. Generation of fundamental data on the basis of replication within the WNV avian reservoir will fill a critical gap in our knowledge of WNV transmission and aid in the improvement of existing surveillance strategies and predictive emergence models for the prevention of human and veterinary disease. The hypothesis that the emergence of WNV within North America has been the result of the introduction of a new viral genotype capable of efficient avian replication will be addressed with the following research aims: (1) Identify WN viral genetic determinants responsible for differential crow virulence through the generation of viral chimeras between avian virulent and avirulent WNV strains. The resulting chimeras will be tested for their ability to produce high viremia and morbidity within crows as well as replicate at elevated temperatures in an in vitro replication system. (2) Elucidate differences in crow pathogenesis between WNVs in order to identify the pathogenic mechanism(s) that underlie differential avian Virulence. Tissue tropism, neuro-invasion and viral persistence will be correlated with specific genetic determinants. Mosquito infectivity will be assessed through the generation of a dose-infection model, allowing for the estimation of the time to which different chimeras will be infectious to mosquitoes. (3) Assess the potential for the emergence of alternative WNV genotypes through the incorporation of the minimal virulence determinants previously identified into alternative WN viral genetic backbones that have not been associated with avian mortality. Adaptation of avirulent WNV genotypes to crows will also be investigated to gauge selection for bird virulence as a mechanism for the emergence of the North American WNV genotype. Critical basic information on the role of viral replication and mechanisms that modulate virulence in natural viral populations within avian reservoir will allow for more efficient implementation of an vaccination programs and the prediction of future WNV outbreaks. In addition, fundamental knowledge of viral replication and pathogenesis will provide insight for the development of vaccine and antiviral strategies. [unreadable] [unreadable]